Transfection of CYP4A1 cDNA increases vascular reactivity inrenal interlobar ar - 《American Journal of Physiology》美国生理学杂志干细胞之家



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发表于 2009-4-21 13:23 |只看该作者 |倒序浏览 |打印

作者：Jun-IchiKaide, Mong-HengWang, Ji-ShiWang, FanZhang, V.RajGopal, John R.Falck, AlbertoNasjletti,  MichalLaniado-Schwartzman作者单位：1 Department of Pharmacology, New York MedicalCollege, Valhalla, New York 10595; and Departmentsof Biochemistry and Pharmacology, University of Texas SouthwesternMedical Center, Dallas, Texas 75235 / O2 H  R3 Z$ v% M
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      【摘要】
   20-HETE, a cytochrome P -450 4A (CYP4A1)-derived arachidonic acid metabolite,is a major eicosanoid formed in renal and extrarenal microcirculation.20-HETE inhibits Ca 2  -activated K channels invascular smooth muscle cells and thereby may modulate vascularreactivity. We transfected renal interlobar arteries with an expressionplasmid containing the cDNA of CYP4A1, the low- K m arachidonic acid -hydroxylase, andexamined the consequences of increasing 20-HETE synthesis onconstrictor responses to phenylephrine. CYP4A1-transfected interlobararteries demonstrated a twofold increase in CYP4A protein levels and20-HETE production compared with arteries transfected with the emptyplasmid; they also showed increased sensitivity to phenylephrine, asevidenced by a decrease in EC 50 from 0.37 ± 0.04 µMin plasmid-transfected arteries to 0.07 ± 0.01 µM inCYP4A1-transfected arteries. The increased sensitivity to phenylephrinewas greatly attenuated by N -methylsulfonyl-12,12-dibromododec-11-enamide (DDMS),a selective inhibitor of 20-HETE synthesis, and by20-hydroxyeicosa-6( Z ),15( Z )-dienoic acid, aspecific 20-HETE antagonist. This effect of DDMS was reversed byaddition of 20-HETE, further substantiating the notion that increasedlevels of 20-HETE contribute to the increased sensitivity tophenylephrine in vessels overexpressing CYP4A1. These data suggest that20-HETE of vascular origin sensitizes renal vascular smooth muscle to phenylephrine. & z: z9 T; K1 B4 B0 V
      【关键词】 hydroxyeicosatetraenoic acid arachidonic acid phenylephrine cytochrome P
   INTRODUCTION

SMALL ARTERIAL VESSELS MANUFACTURE 20-HETE, a product of arachidonic acid metabolism bycytochrome P -450 enzymes of the 4A family (CYP4A1) ( 8, 12, 17, 26 ). Exogenous 20-HETE was reported to contract or relaxvascular smooth muscle, depending on the animal species, type ofvessels, and experimental conditions. Cyclooxygenase-dependent and-independent mechanisms have been implicated in 20-HETE-inducedvascular contraction ( 6, 16, 17 ) and vascular relaxation( 2, 4 ).' u' ^/ ~  v5 Y- F

Renal preglomerular vessels express CYP4A proteins, produce 20-HETE,and are constricted by exogenous 20-HETE. The constrictor action ofexogenous 20-HETE in pressurized canine arcuate arteries, rat renalinterlobular arteries, and rat renal afferent arterioles is independentof cyclooxygenase activity and has been attributed to inhibition of theopening of Ca 2  -activated K channels invascular smooth muscle cells, leading to cellular depolarization andincreased Ca 2  entry ( 8, 12, 14, 23, 28 ).. D  l- f3 P5 S# a( y( O

Recent studies support the notion that endogenous 20-HETE subservesvasconstrictor mechanisms in the rat kidney. For example, inhibitors of20-HETE synthesis were shown to increase blood flow to the kidney( 29 ), to increase the diameter of preconstricted interlobular arteries denuded of endothelium ( 11 ), and toattenuate vasconstrictor responses elicited by endothelin ( 10, 22 ), angiotensin II ( 5 ), or increases in transmuralpressure ( 14 ). 20-HETE produced by the smooth muscleisolated from renal preglomerular vessels was also shown to exert atonic inhibitory influence on the activity of large-conductanceCa 2  -activated K channels ( 28 ).
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Previous studies indicated that the responsiveness of small arterialvessels to constrictor stimuli increased after treatment with a blockerof large-conductance Ca 2  -activated K channels( 13 ). Hence, it is conceivable that 20-HETE of vascular origin is a stimulatory regulator of vascular reactivity to constrictor agonists. To test this hypothesis, we transfected rat renal interlobar arteries with an expression plasmid containing the cDNA of CYP4A1, thelow- K m arachidonic acid -hydroxylase( 20 ), and examined the consequences of enhanced CYP4A1expression on 20-HETE synthesis and vascular reactivity tophenylephrine. We also studied the effect of exogenous 20-HETE and ofagents that interfere with the synthesis or the actions of 20-HETE onvascular reactivity in arterial preparations transfected with plasmidcontaining CYP4A1 cDNA compared with those transfected with the control plasmid.

MATERIAL AND METHODS' W1 R8 _2 d, Z( \
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Transfection of renal interlobar arteries. Full-length CYP4A1 cDNA ( 20 ) was cloned into theexpression vector pcDNA3.1( ) (Invitrogen). The expression plasmidcontaining CYP4A1 cDNA (pcDNA3.1-4A1) and the control plasmid(pcDNA3.1) were encapsulated into liposomes (DOTAP LiposomalTransfection Reagent, Boehringer Mannheim, Indianapolis, IN).Sprague-Dawley rats (8 wk old) were anesthetized with pentobarbitalsodium, the kidneys were perfused with ice-cold Krebs solution, andrenal interlobar arteries were isolated by microdissection under amicroscope. The vessels were placed in culture dishes containing DMEMwith 10% Nu-serum, 100 µg/ml streptomycin, and 100 µg/mlpenicillin plus 17 µg of either pcDNA3.1-4A1 or pDNA3.1. Thevessels were maintained in organ culture for 18 h in a humidifiedincubator (95% air-5% CO 2 ) at 37°C. At the end of theculture period, vessels were used for measurements of CYP4A protein and20-HETE synthesis and for vascular reactivity studies.

CYP4A protein and 20-HETE synthesis. Microdissected arteries were homogenized and proteins were separated byelectrophoresis on an 8% SDS-polyacrylamide gel, transferred to apolyvinylidene difluoride membrane, and blotted with goat anti-ratCYP4A1 polyclonal antibody (1:1,000; Gentest; Woburn, MA).Immunoreactive proteins were detected by phosphorimaging using Vistraenhanced chemifluorescent substrate ( 20 ). 20-HETE production was determined by incubating interlobar arteries with arachidonic acid (30 µM) in 1 ml Tyrode's buffer containing 1 mMNADPH, 10 µM indomethacin, and 10 µM N -nitro- L -arginine methyl esterfor 1 h at 37°C. The reaction was terminated byacidification to pH 3.5-4.0 with 10 µl of 2 M formic acid.[20,20- 2 H 2 ]20-HETE (1 ng) was added as aninternal standard. The mixture was then extracted twice with 2 ml ethylacetate. The final extract was evaporated under nitrogen, resuspendedin 30 µl of methanol, and subjected to reverse-phase HPLC asdescribed ( 18 ). Fractions coeluting with the 20-HETEstandard were collected, evaporated to dryness, and derivatized to thepentafluorobenzyl bromide ester trimethylsilyl ether. 20-HETE wasquantified by negative chemical ionization-GC-MS as previouslydescribed ( 18 ). After extraction, the vessels werecollected and suspended in 100-300 µl of 1 N NaOH, in which theywere left overnight to dissolve. Any intact vessels remaining weremanually ground with a glass rod. Protein concentration was determinedby using the Bio-Rad assay.+ i' r5 \0 r: b& }+ W/ V

Measurement of isometric tension in vascular rings. Interlobar arteries (~230 µm, internal diameter) were cut into ringsegments (2 mm). The rings were mounted on wires in the chambers of amultivessel myograph (JP Trading, Aarhus, Denmark) filled with Krebsbuffer (37°C) and gassed with 95% O 2 -5%CO 2. After 30- to 60-min equilibration, the vessels wereset to an internal circumference equivalent to 90% of that which theywould have in vitro when relaxed under a transmural pressure of 80 mmHg. Isometric tension (mN/mm vessel length) was monitoredcontinuously before and after experimental interventions. Constrictorresponse to 60 mM KCl was determined in each vessel. Subsequently, acumulative concentration-response curve to phenylephrine(10 9 5 × 10 5 M) or to KCl(10 3 10 1 M) was constructed. In someexperiments, the response to phenylephrine and KCl was measured invessels pretreated with N -methylsulfonyl-12,12-dibromododec-11-enamide (DDMS; 30 µM), 20-hydroxyeicosa-6( Z ),15( Z )-dienoic acid(20-HEDE; 10 µM), and 20-HETE (1 or 10 µM) or withtetraethylammonium (TEA; 1 mM). DDMS is a selective inhibitor ofarachidonic acid -hydroxylation ( 25 ). 20-HEDE wasreported to block vascular actions of 20-HETE ( 1 ). TEA isa blocker of large-conductance Ca 2  -activatedK channels.

Data analysis. Data are expressed as means ± SE. Concentration-response dataderived from each vessel were fitted separately to a logistic functionby nonlinear regression. Maximum asymptote of the curve and theconcentration of agonist producing EC 50 were calculated byusing commercially available software (Prism 2.01, GraphPAD software,San Diego, CA). Concentration-response data were analyzed by a two-wayanalysis of variance followed by Duncan's multiple-range test. Otherdata were analyzed by Student's t -test for paired orunpaired observations as appropriate. The null hypothesis was rejectedat P
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RESULTS# \* d) C* I* N, V
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Figure 1 displays data on CYP4Aprotein expression and 20-HETE synthesis by isolated rat renalinterlobar arteries incubated for 18 h with either the expressionplasmid containing CYP4A1 cDNA (pcDNA3.1-4A1) or the controlexpression plasmid (pcDNA3.1). Western blot followed bydensitometry analysis demonstrated that the levels ofCYP4A-immunoreactive proteins in pcDNA3.1-4A1-treated arteries wasincreased by 263 ± 25% (mean ± SE, n = 3)compared with arteries incubated with the control plasmid (Fig. 1 A ). Furthermore, as seen in Fig. 1 B, the rate of20-HETE synthesis in arteries incubated with pcDNA3.1-4A1 was twotimes higher than that in arteries incubated with the control plasmid.
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Fig. 1. CYP4A protein expression and 20-HETE synthesis inrenal interlobar arteries transfected with CYP4A1 cDNA. A :representative immunoblot analysis of CYP4A proteins in isolated renalinterlobar arteries treated with plasmid containing CYP4A1 cDNA(pcDNA3.1-4A1) or empty plasmid (pcDNA3.1) for 18 h asdescribed in MATERIALS AND METHODS. Vessels werehomogenized and proteins were separated on an 8% SDS-polyacrylamidegel, transferred to a polyvinylidene difluoride membrane, and blottedwith goat anti-rat CYP4A1 polyclonal antibody. Immunoreactive proteinswere detected by phosphorimaging methods using Vistra enhancedchemifluorescent substrate as described in MATERIALS AND METHODS. 4A1st, CYP4A1 standard. B : 20-HETE synthesisin renal interlobar arteries transfected with plasmid containing CYP4A1cDNA or empty plasmid. Values are means ± SE; n = 5. * P! @6 H8 X( I$ f4 h; H3 o8 o) s6 h

Figure 2 contrasts renal interlobararteries treated with pcDNA3.1-4A1 or with the control plasmidpcDNA3.1 in terms of constrictor responsiveness to phenylephrine andKCl. In both treatment groups, these agonists elicitedconcentration-dependent increases in isometric tension. Theconcentration-response curve to phenylephrine in vessels treated withpcDNA3.1-4A1 paralleled that in vessels treated with pcDNA3.1 butwas clearly shifted to the left, resulting in a pronounced reduction ofthe EC 50 value without alteration of the maximal response(Fig. 2 A ). In contrast, the concentration-response curve toKCl was nearly identical in vessels treated with pcDNA3.1-4A1 orwith pcDNA3.1 (Fig. 2 B ). Thus transfection of renalinterlobar arteries with CYP4A1 cDNA increased the sensitivity of thevessels to phenylephrine but not to KCl." K$ H! n1 s4 g! v$ a! |

Fig. 2. Concentration-dependent constrictor responses tophenylephrine ( A ) and KCl ( B ) inCYP4A1-transfected renal interlobar arteries. Isolated renal interlobararteries were preincubated with plasmid containing CYP4A1 cDNA or emptyplasmid for 18 h as described in MATERIALS AND METHODS. Vessels were mounted on a wire myograph and changes inisometric tension to increasing concentrations of phenylephrine or KClwere measured as described in MATERIALS AND METHODS. Valuesare means ± SE. * P, c5 I. ~' Y4 ^  B7 J! d

Additional experiments in renal interlobar arteries transfected withthe control plasmid pcDNA3.1 revealed that the EC 50 for phenylephrine (0.33 ± 0.03 µM; n = 6) wasdecreased ( P by treatment with 10 µM 20-HETE(0.08 ± 0.02 µM; n = 6), 1 mM TEA (0.09 ± 0.02 µM; n = 6), or both 20-HETE and TEA combined (0.11 ± 0.03 µM; n = 6) without accompanyingchanges in maximal response (4.32 ± 0.10 mN/mm for controlvessels vs. 4.14 ± 0.09, 4.31 ± 0.09, and 3.78 ± 0.33 mN/mm, respectively, for vessels treated with 20-HETE, TEA, or 20-HETEand TEA combined). On the other hand, the EC 50 forphenylephrine in vessels transfected with pcDNA3.1-4A1 (0.10 ± 0.01 µM; n = 6) was not reduced further byexposure to 10 µM 20-HETE (0.11 ± 0.01 µM; n = 6), 1 mM TEA (0.09 ± 0.01 µM; n = 6), or20-HETE and TEA combined (0.08 ± 0.02 µM; n = 6); neither was the maximal response of these vessels to phenylephrineaffected by the treatments (4.14 ± 0.14 mN/mm for control vesselsvs. 3.98 ± 0.11, 4.10 ± 0.16 and 3.85 ± 0.26 mN/mm,respectively, for vessels exposed to 20-HETE, TEA, or 20-HETE and TEA combined).+ i- h; r$ s  S0 \) W1 Z% P% ~& l  k
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In complimentary experiments, neither 10 µM 20-HETE nor 1 mM TEAaffected the EC 50 for KCl in renal interlobar arteries( n = 6) transfected with the control plasmid pcDNA3.1(13.20 ± 1.27 mM in control vessels vs. 13.74 ± 1.29 and15.59 ± 1.97 mM, respectively, in vessels exposed to 20-HETE andTEA); also, the maximal response to KCl was unaffected by the treatment(3.11 ± 0.22 mN/mm in control vessels vs. 2.88 ± 0.33 and3.19 ± 0.46 mN/mm, respectively, in vessels exposed to 20-HETEand TEA). Similarly, 10 µM 20-HETE and 1 mM TEA were without effecton the EC 50 for KCl in renal interlobar arteries( n = 6) transfected with pcDNA3.1-4A1 (14.19 ± 2.28 mM in control vessels vs. 14.62 ± 2.5 and 13.81 ± 2.19 mM, respectively, in vessels exposed to 20-HETE and TEA); also, the maximal response to KCl was unaltered by the treatments (3.23 ± 0.25 mN/mm in control vessels vs. 2.90 ± 0.31 and 3.17 ± 0.39 mN/mm, respectively, in vessels exposed to 20-HETE and TEA)./ \6 d# b: P% s4 ]9 d- u

As shown in Fig. 3, the increasedsensitivity to phenylephrine in renal interlobar arteries transfectedwith CYP4A1 cDNA was greatly attenuated by agents that inhibit thesynthesis or action of 20-HETE. DDMS, a selective inhibitor ofCYP4A-catalyzed arachidonic acid -hydroxylation ( 25 ),caused a rightward shift in the concentration-response curve tophenylephrine in pcDNA3.1-4A1-treated vessels, increasing theEC 50 by ~10-fold without affecting the maximal response(Fig. 3 A ). 20-HEDE, a putative antagonist of 20-HETE( 1 ), also caused a rightward shift in theconcentration-response curve to phenylephrine in vessels treated withpcDNA3.1-4A1, increasing the EC 50 by about eightfoldwithout affecting the maximal response (Fig. 3 A ). On theother hand, in interlobar arteries treated with the control plasmidpcDNA3.1, DDMS had no effect, whereas 20-HEDE slightly increased theEC 50 (Fig. 3 B ). Thus DDMS and 20-HEDE areeffective in attenuating the sensitivity to phenylephrine in vesselstreated with pcDNA3.1-4A1, which express higher levels of CYP4A1protein and manufacture greater amounts of 20-HETE. In complimentaryexperiments, DDMS was without effect on the EC 50 for KCl inrenal interlobar arteries transfected with pcDNA3.1(13.20 ± 1.27 vs. 12.90 ± 1.40 mM; n = 6)or pcDNA3.1-4A1 (14.19 ± 2.28 vs. 14.32 ± 1.93 mM; n = 6); DDMS also was without effect on the maximalresponse to KCl (3.11 ± 0.22 vs. 3.04 ± 0.26 mN/mm invessels transfected with pcDNA3.1 and 3.23 ± 0.25 vs. 3.29 ± 0.38 mN/mm in vessels transfected with pcDNA3.1-4A1).; p! c4 }* v" o  c( I+ b( o
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Fig. 3. Effect of N -methylsulfonyl-12,12-dibromododec-11-enamide (DDMS) and20-hydroxyeicosa-6( Z ),15( Z )-dienoic acid(20-HEDE) on concentration-dependent constrictor responses tophenylephrine in sham- and CYP4A1-transfected renal interlobararteries. Isolated renal interlobar arteries were preincubated withplasmid containing CYP4A1 cDNA ( A ) or with empty plasmid( B ) in MEM containing 10% FBS for 18 h as described in MATERIALS AND METHODS. Arteries were mounted on a wiremyograph, and changes in isometric tension to increasing concentrationsof phenylephrine in the presence of DDMS (30 µM) or 20-HEDE (10 µM)were measured as described in MATERIALS AND METHODS. Valuesare means ± SE, number of experiments for each treatment isshown. * P  s( z. L: N5 i1 `

If inhibition of 20-HETE synthesis is responsible for the desensitizingeffect of DDMS on phenylephrine-induced contraction of vesselstransfected with CYP4A1 cDNA, the inclusion of exogenous 20-HETE intothe bathing buffer may be expected to offset the DDMS-inducedattenuation of vascular reactivity in such vessels. Figure 4 illustrates the results of experimentsexamining the effect of 20-HETE on constrictor responsiveness tophenylephrine in CYP4A1-tranfected interlobar arteries bathed in buffercontaining and not containing DDMS. In vessels in which treatment withDDMS had produced a rightward shift in the concentration-response curve to phenylephrine, exogenous 20-HETE negated the DDMS-inducedattenuation of vascular reactivity, partially at 1 µM (Fig. 4 A ) and completely at 10 µM (Fig. 4 B ). As notedabove, 20-HETE did not affect any aspect of theconcentration-response curve to phenylephrine in CYP4A1-transfected vessels not exposed to DDMS (Fig. 4, A and B ).
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Fig. 4. Effect of 20-HETE on constrictor responses tophenylephrine in CYP4A1-transfected renal interlobar arteriespretreated and not pretreated with DDMS. Isolated renal interlobararteries preincubated with the pcDNA3.1-4A1 for 18 h weremounted on a wire myograph. Changes in isometric tension produced byincreasing concentrations of phenylephrine in preparations with andwithout DDMS (30 µM) pretreatment were measured in the absence andpresence of 1 ( A ) or 10 µM 20-HETE ( B ). Valuesare means ± SE. * P* ?; h, ~7 F! f. ]9 d

DISCUSSION. S7 B5 n3 @& v' a, \

Successful transfer of genes into cells and tissues by means ofviral and nonviral vectors is well documented. In the present study, weused a nonviral approach to achieve overexpression of CYP4A1, anarachidonic acid -hydroxylase, which catalyzes the synthesis of20-HETE with an efficiency 10 and 40 times greater than that of CYP4A2and CYP4A3, respectively ( 20 ). Rat renal interlobararteries cultured for 18 h in media supplemented with anexpression plasmid containing CYP4A1 cDNA under the control of theCMV promoter displayed greater CYP4A protein levels and a higher rateof 20-HETE synthesis than vessels cultured in media supplemented withan expression plasmid not containing CYP4A1 cDNA. That CYP4A proteinexpression and 20-HETE synthesis are increased in vessels treated withCYP4A1 cDNA-containing plasmid is indicative of successful transfectionleading to enhanced vascular expression of functional CYP4A1.
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Renal interlobar arteries transfected with CYP4A1 cDNA differedstrikingly from nontransfected vessels in terms of contractile responsiveness to phenylephrine. In the CYP4A1-transfected vessels, theconcentration-response curve to phenylephrine was shifted to the leftso that the EC 50 value was reduced, whereas the maximal response was unchanged. These findings imply that the vascular sensitivity to phenylephrine is increased in CYP4A1-transfected vessels. This increase in sensitivity is not generalized, because thecontractile responsiveness to KCl was not affected in vessels treatedwith CYP4A1 cDNA.$ f7 d: O$ x: d$ V

According to our studies, both the CYP4A inhibitor DDMS and the 20-HETEantagonist 20-HEDE were effective in offsetting the enhancedsensitivity to phenylephrine displayed by CYP4A1-transfected renalinterlobar arteries. These observations are compelling evidence thatthe augmented production of 20-HETE brought about by increased expression of CYP4A1 is responsible for sensitizing these vessels tophenylephrine. Such a regulatory action of endogenous 20-HETE isconsistent with the finding that exogenous 20-HETE elicits concentration-dependent sensitization to phenylephrine inCYP4A1-transfected vessels pretreated with DDMS to inhibit endogenous20-HETE synthesis.

Sensitization to phenylephrine by exogenous 20-HETE was notdemonstrable in CYP4A1-transfected vessels not pretreated with DDMS.Conceivably, under such circumstances, the vascular production of20-HETE is already sufficiently high to achieve maximal sensitization to the agonist. In vessels pretreated with an empty plasmid not containing CYP4A1 cDNA, the sensitivity to phenylephrine was not significantly reduced by DDMS and was minimally attenuated by 20-HEDE.One likely interpretation of these observations is that the basalproduction of 20-HETE under such circumstances is below that requiredfor promoting sensitization to phenylephrine. Collectively, ourfindings support the concept that 20-HETE of vascular origin is astimulatory regulator of vascular reactivity to phenylephrine.
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A relatively high concentration of 20-HETE, 10 µmol/l, is required tobring up the sensitivity to phenylephrine displayed by control vesselsnot overexpressing CYP4A1, or by CYP4A1-transfected vessels treatedwith DDMS, to the level of sensitivity in interlobar arteriesoverexpressing CYP4A1. This may be indicative of differences inmetabolic disposition and accessibility to target sites between exogenous and endogenous 20-HETE. For example, the access of exogenous 20-HETE to potential sites of action, that is, cellular binding sitesor receptors ( 1 ), protein kinase C ( 15, 21, 24 ), and mitogen-activated protein kinase(s) ( 24, 19 ), may be limited by further metabolism ( 9, 16 ),esterification into cellular lipids ( 3 ), or interactionwith extracellular structures.- |- ]; J7 ?( f5 o

Previous studies have demonstrated that the activity oflarge-conductance Ca 2  -activated K channels inrenal preglomerular vascular smooth muscle cells is decreased byexogenous 20-HETE and increased by CYP4A inhibitors ( 28 ).Similar effects were observed in the cerebral microcirculation ( 7, 15 ). On the basis of such findings, it has beenproposed that 20-HETE of vascular origin is an inhibitory regulator of the Ca 2  -activated K channels in vascularsmooth muscle. Inhibition of these channels is expected to favordepolarization and contractile responsiveness of vascular smoothmuscle. Indeed, in our study, TEA pretreatment increased thesensitivity to phenylephrine in renal interlobar arteries notoverexpressing CYP4A1. Thus the sensitizing action of 20-HETE onphenylephrine-induced vascular contraction may be a consequence of theinhibitory action of this eicosanoid on the activity of thelarge-conductance Ca 2  -activated K channels.It is conceivable that 20-HETE production in vessels transfected withpcDNA3.1-4A1 is sufficiently increased to maximally suppress theactivity of such channels. This would be consistent with our findingthat neither exogenous 20-HETE nor TEA, alone or in combination, waseffective in sensitizing vessels overexpressing CYP4A1 to phenylephrine.

In summary, this study documents overexpression of CYP4A and increased20-HETE synthesis in rat renal interlobar arteries maintained inculture for 18 h in media that includes an expression plasmidcontaining CYP4A1 cDNA. The sensitivity of interlobar arteries treatedwith CYP4A1 cDNA expression plasmid to phenylephrine greatly exceededthat of vessels treated with a plasmid not containing CYP4A1 cDNA. Aninhibitor of CYP4A, DDMS, as well as an antagonist of 20-HETE actions,20-HEDE, were effective in offsetting the increased sensitivity tophenylephrine brought about by CYP4A overexpression. These observationssuggest that increased synthesis of 20-HETE in vessels transfected withCYP4A1 cDNA is responsible for their sensitization to phenylephrine.The study calls attention to the possibility that 20-HETE produced byarterial vessels contributes to vasoconstrictor mechanisms bysensitizing the vasculature to the action of constrictor agonists insituations wherein there is increased vascular CYP expression and/oractivity. Relative to this point, a recent study documented a role for20-HETE in the increased sensitivity of small mesenteric arteries ofspontaneously hypertensive rats to phenylephrine and vasopressin( 27 ).
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ACKNOWLEDGEMENTS" L4 x" Y# }$ j8 f6 K6 ?3 d# u

This study was supported by National Institutes of Health GrantsPO1-HL-34300, RO1-HL-18579, and GM-31278, American Heart AssociationNew York Affiliate Grants 0020152T and 99-30277T, and the RobertA. Welch Foundation.$ o0 o* c4 A' ~' F
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